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Development of new in situ hybridization method using freeze transfer.

使用冷冻转移开发新的原位杂交方法。

基本信息

批准号：
06557094
负责人：
KURISU Kojiro
金额：
$ 3.9万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557094/
关键词：
Gene expression mRNA PCR method Nitrocellulose memnrane Cryosection RNA probe Digoxygenin in situ hybridization 冷凍切片 in situ ハイブリダイゼーションヒトロセルロース膜 in sutu ハイブルダイゼーション

项目摘要

In situ hybridization (ISH) is an useful technique to localize gene expression in a tissue section. Its time consuming and complex procedure requires great skill of researchers. In the present study, we developed simpler and sensitive ISH method by mean of freeze transfer technique.Limbs and heads, obtained from 14-day embryonic mouse, were frozen and embedded in OCT compound. Frozen sections (8mum thick) were mounted directly onto the surface of nitrocellulose membrane and were quickly dried by hair drier. Membranes were then treated with 10mug/ml protenase K for 10-40- min, refixed with 4% PFA,and hybridized with digoxigenin-labelled type I,II,X collagen, and qggrecan riboprobes. In a parallel experiment, conventional ISH were carried out with same tissue sections and probes.In all experiments, we obtained higher signal and lower back-ground by our new ISH.This technique was superior to a conventional ISH in following points. Once the tissue section were mounted on the membrane, tissue sections were not peeled off from the membrane even after 70ﾟC hybridization and 70ﾟC wash. We could omit acetylation and RNase treatment step which is usually necessary to reduce back-ground. In addition, our new ISH required only 1/10-1/100 of probe amount which was used in a conventional ISH.Therefore our new ISH is technically simpler and more sensitive than conventional ISH.

原位杂交（ISH）是一种在组织切片中定位基因表达的有用技术。其耗时和复杂的过程需要研究人员的巨大技能。本研究利用冷冻转移技术建立了一种简便、灵敏的原位杂交方法，取14天胚胎小鼠的四肢和头部，冷冻后包埋在OCT复合物中。将冷冻切片（8 μ m厚）直接固定在硝酸纤维素膜的表面上，并通过吹风机快速干燥。然后用10 μ g/ml蛋白酶K处理膜10-40分钟，用4%PFA再固定，并与地高辛标记的I、II、X型胶原和qggrecan核糖探针杂交。在一个平行的实验中，我们用同样的组织切片和探针进行了常规ISH，在所有的实验中，我们用我们的新ISH获得了更高的信号和更低的背景。一旦将组织切片固定在膜上，即使在70 ° C杂交和70 ° C洗涤后，组织切片也不从膜上剥离。我们可以省略乙酰化和RNA酶处理步骤，这通常是减少背景所必需的。此外，我们的新型ISH所需探针量仅为常规ISH的1/10-1/100，因此，我们的新型ISH比常规ISH在技术上更简单，灵敏度更高。